The invention lies in the field of diagnostics. More in particular, the invention lies in the field of molecular diagnostics.
The increased knowledge of the molecular basis for disease has generated an increasing demand for more and more sophisticated diagnostic methods that can help identify the exact molecular cause of disease. In particular, for infectious diseases, clinicians want to be able to rapidly identify the pathogen. Importantly, concurrent accurate typing and discrimination of different strains of a pathogen is desired. This is important, for instance, in cases where certain strains have a particular unfavorable phenotype. It is also important, for instance, in the case where the pathogen is capable of rapid mutation of its genome to counteract selective pressures induced by the patient and/or the treatment. One non-limiting example of such a pathogen is, of course, Human Immunodeficiency Virus (xe2x80x9cHIVxe2x80x9d). HIV is, for instance, capable of evading selective pressure induced by nucleotide analogues through mutation of the reverse transcriptase enzyme. To be able to predict which nucleotide analogue, if any, would benefit the patient, it is desired to know in advance, i.e. before treatment starts, which genotype(s) of HIV prevail in the patient.
One possibility to find the pathogen causing the disease is to harvest a sample from the patient comprising the pathogen and culturing the pathogen on suitable media in the case of a bacterial pathogen or in a suitable marker cell line for a viral and/or mycobacterium pathogen. The pathogen may be typed following and/or during culturing. This culture process can be combined with, for instance, antibiotics and/or other medicines to find the relative resistance/sensitivity of the pathogen to said medicine. This so-called culture driven testing has several advantages and is indeed routinely applied for a number of diseases.
However, generally, the considerable amount of time involved with the culture process necessitates that a treatment schedule be started prior to the identification of the causative agent. This is not desired, since the treatment started may prove to be ineffective. Moreover, for many pathogens, a culture system is as yet not available. Another problem with the culture system is the inherent variability of the procedure. Not all pathogens are equally well cultured outside the body of a patient. In addition, since viability of the pathogen is essential, differences in the handling of the sample outside the body will result in variability of the result. Moreover, the costs involved in the initiation of a screen with the culture system for a wide variety of different possible causative agents in any clinical sample are considerable.
For this reason there is a need for a rapid system for the typing of a pathogen that is versatile, reliable and at least partially able to discriminate between different variants of the pathogen. A number of different strategies have been tried. One such strategy relies on the detection of pathogen-derived nucleic acid in a sample. To be able to rapidly detect such nucleic acid, a nucleic acid amplification step is usually required.
Many nucleic acid amplification methods have been devised that are able to specifically detect a certain pathogen and possibly even a number of different strains of said pathogen. However, such methods usually require the clinician to have at least some idea of the kind of pathogen that may cause the disease in the patient. This is frequently not the case.
The present invention provides a method for detecting, quantifying and/or simultaneous typing of a variety of different nucleic acid sequences in a sample. Preferably, said nucleic acid sequences comprise nucleic acid from a microorganism and/or derivative thereof. The microorganism can be a bacterium, a phage and/or a virus.
Preferably, said microorganism is a pathogenic microorganism. The present invention further allows discrimination between different strains of a microorganism or other sequences. The present invention is not only useful for the typing of a pathogen in a sample of a patient, but it is also applicable for the typing of a pathogen in a sample derived from an animal. Preferably, said animal has a commercial and/or emotional value to a human, such as a pet, a farm animal and/or an animal living in a natural reserve. The invention is also suitable for application in poultry and fish. The method of the invention is, of course, not only suited for the typing and/or detection of a pathogen. The method is generally suited for the typing and/or detection of nucleic acid in a sample. For instance, in the case of cellular DNA or RNA, the method can be used for creating a genetic expression profile, respectively, of the nucleic acid in the sample. Knowing the origin of the nucleic acid in the sample then allows the correlation of the profile with the origin.
In case the origin is nucleic acid of (a specific part of) an individual, the profile, or a part thereof, can be correlated to, for instance, a database of profiles, or parts thereof, of other individuals. Matching of the profile (or part thereof) to known profiles(or parts thereof) allows the correlation of the profile (or part thereof) of the individual with the phenotypes of individuals with matching profiles(or parts thereof) displayed by these other individuals. Thus, the method of the invention can be used generally for the typing and/or detection of nucleic acid in a sample.
In one aspect, the invention provides a method for amplifying nucleic acid in a sample comprising providing said sample with a set of primers comprising between 3 and 8 random bases and at least 8 essentially non-random bases, subjecting said sample to a first nucleic acid amplification reaction, providing said sample with at least one second primer comprising at least 8 bases essentially identical to said non-random bases, subjecting said sample to a second amplification reaction and detecting nucleic acid amplified in said sample. Typically only a limited amplification of nucleic acid will occur in said first nucleic acid amplification reaction. Said first and second amplification reactions are preferably performed separately, optionally including a step to remove any unused primer in said first amplification reaction. This way, the reproducibility of the method is best controlled. However, the first and the second amplification reactions may also be performed simultaneously.
Nucleic acid in said sample may be DNA and/or RNA. A double stranded nucleic acid can be denatured into essentially single stranded nucleic acid prior to the priming of synthesis of a complementary strand of nucleic acid. The complementary strand may be DNA and/or RNA. Synthesis of said complementary nucleic acid is performed under conditions and using enzymes that are known in the art, such as, for instance, conditions and enzymes commonly used for polymerase chain reaction and/or NASBA.
The number of nucleic acids amplified with the method of the invention is dependent on the amount and the complexity of the nucleic acid in the sample. When the complexity, i.e. the number of different sequences in the nucleic acid(s) is low, a small number of nucleic acids will be amplified with the method of the invention. In this case, some nucleic acids will be dominant in the amplificate, resulting in a banding pattern when the amplificate is run on a gel. On the contrary, when the complexity of the nucleic acid in the sample is high, many nucleic acids will be amplified, resulting in a smear when the amplificate is run on a gel. An example of nucleic acid with a particularly low complexity is nucleic acid derived from a small virus and/or plasmid (typically smaller than 10 kb). An example of nucleic acid with a particularly high complexity is cellular DNA (typically comprising  greater than 108 kb). It is clear that the mentioned examples are non-limiting. Many different complexities are possible, and additionally mixtures of low and high complexity nucleic acid can be used for the present invention. However, samples comprising only nucleic acid of low complexity that is smaller than 1 kb are not suited for the present invention. As mentioned above, the number of nucleic acids effectively amplified with the method of the invention is also dependent on the amount of nucleic acid in the sample. When the sample comprises particularly low amounts of nucleic acid, some nucleic acids will be dominant in the amplificate, resulting in a banding pattern when the amplificate is run on a gel. On the other hand, when the amount of nucleic acid in the sample is high, many nucleic acids will be amplified, resulting in a smear when the amplificate is run on a gel.
The dependencies on the complexity and the amount of nucleic acid in the sample are intertwined. For example, when the complexity of the nucleic acid in the sample is high, but the amount of nucleic acid in the sample is low, some nucleic acids will be dominant in the amplificate. As will be discussed in more detail later in the description, amplificates comprising dominant nucleic acids and amplificates comprising many different nucleic acids and amplificates comprising both dominant and many different nucleic acids are useful in the present invention. However, the present invention is only useful when at least two nucleic acids are amplified. Preferably, at least 5 nucleic acids are amplified. More preferably, at least 50 nucleic acids are amplified. Typically, the method is used to amplify approximately 10,000 different nucleic acids, for instance, in samples comprising a relatively large amount of complex nucleic acid.
Although the dependencies mentioned above are intertwined, the person skilled in the art will be able to determine what amount of nucleic acid is required to obtain a specific resulting amplificate.
Preferably, said sample is provided with a set of primers comprising at least three or more different primers. Preferably, said set comprises at least ten different primers.
A nucleotide may be an A, T, C, G, or a U and/or a functional equivalent thereof. A functional equivalent of a nucleotide is any substance capable of mimicking, at least in part, an A, T, C, G, or a U in a nucleic acid. For instance, known nucleotide analogues are suitable substances. Also, substances that can mimic a couple of nucleotides, such as, for instance, inosine are suitable substances. Preferably, said nucleotide analogues allow continued synthesis of the nascent strand.
As used herein, the term xe2x80x9crandom basexe2x80x9d means that between any two primers in said set of primers, there is at least one nucleotide or a functional equivalent thereof at a certain position that is different between the two primers.
Apart from the random bases, primers in said set of primers further comprise an essentially non-random number of bases. This has the advantage that for subsequent amplification and/or detection of synthesized nucleic acid, an essentially known template is provided such that one or more new primers can be devised that can be used for the subsequent amplification and or detection of said synthesized nucleic acid. Preferably, said essentially non-random number of bases comprises between 17 and 22 nucleotides. Subsequent amplification is typically performed with at least one primer comprising a sequence essentially identical to a sequence formed by non-random bases in said at least one primer. There can, of course, be more than one second primer. Furthermore, a second primer can comprise nucleotides in addition, to the nucleotides required to create identity to a non-random sequence of a primer in the first set of primers. Additional nucleotides at the 3xe2x80x2 end can be advantageously used in applications wherein additional specificity is required in the amplified product. Additional nucleotides at the 5xe2x80x2 end can be advantageously used for the introduction of restriction enzyme sites that can be utilized to clone amplified nucleic acid. Cloning of amplified nucleic acid is often desired when amplified nucleic acid needs to be sequenced.
In the present invention, the number of random bases in the set of primers has been observed to be of crucial importance to the practical application of the method of the invention, for instance, for the detection, quantification and/or typing of nucleic acid in the sample. This is especially crucial to the detection of nucleic acid from a wide variety of different pathogens. When less then 3 random bases are used in the set of primers, the subsequent amplification is not sufficiently versatile to detect a wide variety of different nucleic acids (nucleic acid with different sequences), such as from a wide variety of different microorganisms. Presumably, this is due to a lack of hybridization capability among the various nucleic acids. When more than 8 random bases are used in the set of primers, the signal detected is too specific for particular nucleic acids. When the method is used for the detection of a microorganism, such as a pathogen, this leads to the situation that nucleic acid of the microorganism present in the sample may not be detected with sufficient sensitivity. This is presumably due to the fact that not all possible combinations of 9-mers can be included in a practical way in the amount of primer that can be used in the method of the invention. Without being bound by theory, it is the observation in the present invention that for the capability to detect a wide variety of different nucleic acids, it is necessary to have in the set of primers between 3 and 8 random bases. Preferably, said set of primers comprises between 4 and 7 random bases. More preferably, said set of primers comprises 5 or 6 random bases.
To increase the specificity of the reaction, said random bases are preferably clustered at the 3xe2x80x2 end of the primer. In the present invention, it has been observed useful for optimal yield of amplificate to include a G at the extreme 3xe2x80x2 end of the oligonucleotides of the set of primers. A set of primers of the invention therefore preferably comprises a G at the extreme 3xe2x80x2 end of at least most, and preferably all, of the oligonucleotides contained in the set of primers.
In a preferred embodiment of the invention, the non-random bases in the set of primers comprise a sequence enabling non-nucleic acid-primed nucleic acid synthesis. Such a sequence may be used to obtain further amplification of complementary nucleic acid, which further amplification strengthens the signal obtained from the method of the invention. Moreover, the further amplification may be used in a method tbr determining at least part of a sequence of amplified nucleic acid such that amplified nucleic acid may be typed and/or variants of different nucleic acids, such as different variants and/or strains of a microorganism, may be determined. Preferably the non-nucleic acid-primed nucleic acid synthesis comprises transcription.
In a preferred embodiment, said set of primers comprises the sequence:
5xe2x80x2-GCT ATC ATC ACA ATG GAC NNN NNG-3xe2x80x2 (SEQ ID NO:1), and/or
5xe2x80x2-AAT TCT AAT ACG ACT CAC TAT AGG GNN NNN G-3xe2x80x2 (SEQ ID NO:2),
wherein N can be any nucleotide or functional equivalent thereof.
For the detection of a wide variety of different nucleic acids, such as from different microorganisms, pathogens and/or different variants of a particular microorganism, it is essential that the amplificate of the amplification reaction be scrutinized. This can be done through detecting the amplificate with a probe specific for amplified nucleic acid, for instance, a probe specific for nucleic acid of a microorganism, such as nucleic acid from a pathogen and/or variant of said pathogen. Alternatively, the amplificate is at least in part sequenced, wherein the resolved sequence is specific for nucleic acid of said pathogen and/or variant of said pathogen.
Sequencing of at least part of the amplificate is particularly favorable when the complexity of the nucleic acid in said sample is relatively small, particularly when said sample comprises essentially one type of nucleic acid, such as nucleic acid from one microorganism. However, sequencing of at least part of the amplificate is also possible when the sample comprises two, three or more types of nucleic acid. In this embodiment of the invention, however, the sample preferably does not comprise more than 5 different types of nucleic acid in a substantial amount. In one embodiment of the invention, it is possible with this method to obtain a complete, or at least nearly complete, sequence of a particular nucleic acid present in said sample. A low complexity of the nucleic acid in the sample can be obtained in various ways, for instance, in applications wherein the nucleic acid of, for instance, a microorganism, preferably a virus and/or a phage, is collected into an enriched fraction. For instance, a sample of cell free serum obtained from an HIV-infected patient will be enriched for nucleic acid of HIV viruses. Such samples, or parts thereof, may be used in a method of the present invention. A sequence of an HIV virus present in said sample can then be determined by sequencing of the amplificate obtained with the method of the invention. Furthermore, sequencing of the amplificate will also enable the typing of at least the dominant HIV variants in the sample.
Alternatively, a sequence may be generated representing a gross average of the various variants of HIV in said sample. For this embodiment of the invention, a sample comprising a low complexity of nucleic acid is preferred. A low complexity of nucleic acid in the sample does not mean that said sample may not contain complex nucleic acid, such as cellular DNA. It can contain complex nucleic acid as long as the amount (by weight) of complex nucleic acid does not exceed the amount (by weight) of said low complexity nucleic acid. Preferably, the amount of complex nucleic acid does not comprise more than 25% of the nucleic acid in the sample. More preferably, the amount of complex nucleic acid does not comprise more than 10% of the nucleic acid in the sample. Of course, it is clear to the person skilled in the art that this feature of the present invention is not only useful for the sequencing and/or typing of different HIV variants, but it is also generally applicable for the typing of nucleic acid in said sample.
In another embodiment of the invention, said detecting of amplified nucleic acid comprises subjecting at least part of said amplified nucleic acid to a hybridization reaction with a multiplicity of nucleic acids preferably present in a microarray and/or DNA-chip and detecting whether amplified nucleic acid hybridized with one or more nucleic acids of said multiplicity of nucleic acids. This embodiment is particularly useful when the complexity of the nucleic acid in the sample is relatively large. This embodiment is also very useful when the type of nucleic acid present in said sample is not known. Preferably, the multiplicity of nucleic acids comprise microorganism nucleic acid or nucleic acid that is a reflection of nucleic acid expressed by a cell. The cell may be any type of cell. When the multiplicity of nucleic acid comprises a reflection of nucleic acid expressed by a cell, it is preferred that the nucleic acid in said sample comprises RNA that is or was expressed by a cell. In such a case, the RNA is preferably first transcribed into DNA with, for instance, a primer capable of recognizing the poly-A tail of mRNA.
The method of the invention can further comprise one or more additional amplification reactions using one or more other primers. Such an additional amplification reaction can be advantageously used to pre-amplify xe2x80x9ccertainxe2x80x9d nucleic acid in the sample. Alternatively, an additional amplification reaction can be used to further amplify at least part of the amplificate of said first and/or second amplification reactions. In this embodiment, therefore, a method of the invention is provided, further comprising an additional nucleic acid amplification of nucleic acid in said sample using at least one primer comprising essentially non-random bases.
In one embodiment, the invention provides a set of oligonucleotides comprising a sequence:
5xe2x80x2-GCT ATC ATC ACA ATG GAC NNN NNG-3xe2x80x2 (SEQ ID NO:1), and/or
5xe2x80x2-AAT TCT AAT ACG ACT CAC TAT AGG GNN NNN G-3xe2x80x2 (SEQ ID NO:2),
wherein N can be any nucleotide or functional equivalent thereof. In these sets of oligonucleotides, N delineates the position of a random base and C, A, T and G the position of a non-random base.
In another aspect, the invention provides the use of a set of oligonucleotides and/or primers of the invention for the preferred amplification of at least part of a viral nucleic acid. Preferably said set of oligonucleotides and/or primers comprises between 3 to 8 random bases clustered around the 3xe2x80x2 end of said oligonucleotides and/or primers and an essentially constant sequence at essentially the 5xe2x80x2 end of said oligonucleotides and/or primers for priming the synthesis of a complementary nucleic acid in a nucleic acid amplification method. Typically, said set of oligonucleotides and/or primers provide one or more essentially constant templates for detection and/or further amplification of said complementary nucleic acid. Preferably, said set of primers and/or oligonucleotides comprises a sequence:
5xe2x80x2-GCT ATC ATC ACA ATG GAC NNN NNG-3xe2x80x2 (SEQ ID NO:1), and/or
5xe2x80x2-AAT TCT AAT ACG ACT CAC TAT AGG GNN NNN G-3xe2x80x2 (SEQ ID NO:2),
wherein N can be any nucleotide or functional equivalent thereof. In these sets of oligonucleotides, N delineates the position of a random base and C, A, T and G the position of a non-random base. Preferably, said set of primers provides one essentially constant template for detection and/or further amplification of said complementary nucleic acid.
In yet another aspect, the invention provides the use of a set of primers comprising between 3 and 8 random bases clustered around the 3xe2x80x2 end and one or more essentially constant sequences clustered at essentially the 5xe2x80x2 end of each primer in said set of primers in a nucleic acid amplification reaction comprising nucleic acid for providing complementary nucleic acid generated with said set of primers in said amplification reaction with one or more tags enabling further amplification and/or detection of said complementary nucleic acid.
In yet another aspect, the invention provides a kit for the amplification of nucleic acid in a sample comprising at least one random primer comprising between 3 and 8 random bases. Preferably, said kit comprises at least one set of oligonucleotides and/or primers of the invention. Preferably, said set of primers and/or oligonucleotides further comprise one or more essentially constant sequences clustered at essentially the 5xe2x80x2 end of each primer in said set of primers. Preferably, said nucleic acid in a sample comprises nucleic acid from a microorganism or a derivative thereof.
In yet another aspect, the invention provides the use of a kit of the invention in a method of the invention.